In HIV infection, virus-specific cytotoxic I cell kC I L) response can be readily elicited by stimul ion in vitro. Despite this, viral production continues at a high level in most untreated subjects. Thus, a critical evaluation of the functional response in vivo is required to understand their relevance to host resistance and disease progression. Using the highly sensitive MHC/peptide tetramer staining to detect viral-specific CD8 T cells, we have found that HIV-specific CTL are functionally defective in chronically infected subjects in vivo. However, tetramer staining allows the study of only one of a gamut of epitopes involved in the immune response. Using a novel assay which measures function after challenge with HIV-infected primary CD4 T cell targets, we were able to detect some functional cells. Further preliminary data suggest that although virus! infected cells are expected to present all viral antigens, the functional response is directed only against few t epitopes. In aim 1, we will test the hypothesis that the functional epitopes are immunodominant in vivo and the non-functional epitopes represent subdominant epitopes that are not actively involved in the immune response. We will characterize the phenotype and epitope recognition of CD8 T cells that produce IFN-gamma in response to HIV-infected primary CD4 T cells and compare them to the total body of HIV epitopes identified using peptide pools and a panel of MHC/peptide tetramers. In aim 2, we will analyze the mechanisms that lead to an accumulation of non-functional HIV-specific CD8 T cells. One of the viral proteins, nef is known to down regulate the key antigen presenting molecule, class I MHC. To study the role of nef, we will use nef+ or nef- virus to infect CD4 T cells and use these stimulator cells to compare the CD8 T cell response. Another prominent feature of HIV is its ability to mutate rapidly to evade immune response. Because the antigen is no longer presented after mutation, the original immunodominant epitope may become irrelevant, and hence subdominant. To test this possibility, we will compare the clonal composition of immunomagnetically-captured IFN-gamma producing T cells responding to the lab adopted HIVIIIB virus vs patients' own (autologous) virus-infected CD4 T cell targets by analyzing their T cell receptor repertoire. We will also test if the functional cells responding to HIVIIIB virus can recognize autologous virus and conversely, whether cells responding to autologous virus can recognize HIVIIIB virus infected targets.